Nucleic and amino acid sequences for the control of pathogen agents

ABSTRACT

The present invention reveals a nucleic acid sequence from  Nicotiana megalosiphon  encoding for an anti-pathogenic protein. The invention comprises the use of this nucleic acid molecule in transgenic plants of agricultural interest to confer resistance to pathogens. The invention also includes a bioproduct that comprises this anti-pathogenic protein to control plant pathogen agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/934,685filed on Sep. 27, 2010, now U.S. Pat. No. 8,450,559, which is a NationalPhase application of International Application No. PCT/CU2009/000003filed Mar. 3, 2009, which claims priority based on Cuban Application No.2008-0045 filed Mar. 28, 2008, which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is related with the field of the agriculturalbiotechnology, specifically with the use of an anti-pathogenic proteinin the control of pathogen agents. When the anti-pathogenic protein isapplied, either by its expression in genetically modified plants, or asa bioproduct, high control levels of the diseases produced by pathogenagents of plants are obtained.

DESCRIPTION OF RELATED ART

The plant anti-pathogenic proteins have been isolated from leaves,sheaths, roots, tubers, shafts, fruits and flowers, from crop as radish,onion, medic, pepper, potato and soja. The anti-pathogenic proteins havebeen studied at biochemical and structural level, they are smallpeptides, of approximately 5 KDa, with 45 to 54 amino acids, rich incysteines, highly basic, and charged positively. The family of theanti-pathogenic proteins of plants is diverse with regard to the aminoacids composition, since only the eight cysteines that stabilize thestructure seem to be conserved. This characteristic shows the diversebiological activities exhibited by the different plant anti-pathogenicproteins (Broekaert et al. (1995) Plant Physiol. 108:1353-8). The plantanti-pathogenic proteins can be divided in two groups, according to thestructure of the protein precursor; in the first group the proteinprecursor is composed by a signal peptide from endoplasmic reticulum anda mature protein domain. This protein enters into the secretor ways andhas not signals for the post-transcriptional process. In the secondgroup, the anti-pathogenic protein is formed by a long precursor thatcontains besides the signal peptide and the mature domain, a C-terminalpre-domain of 33 amino acids approximately. So far, theseanti-pathogenic proteins have been found only in Solanaceae species (Layy Anderson (2005) Curr Protein Pept Sci. 6:85-101). Not all the plantanti-pathogenic proteins have the same action, some exhibit a potentactivity in vitro against a wide spectrum of filamentous fungus inmicromolar concentrations; others do not inhibit the fungus growth, butinhibit the α-amylase and proteins of synthesis (Colilla et al (1990)FEBS Lett. 270: 191-194).

The model Shai-Matsuzaki-Huang explains the activity of most of theanti-pathogenic proteins, which explains the interaction of the peptidewith the plasmatic membrane followed by a lipidic displacement causesthe formation of multimeric pores inside the plasmatic membrane due tothe insertion of the positively charged protein in the cellular membraneafter the occurrence of its interaction with the negatively chargedfosfolipidic of the target cell surface, these multimeric poresconstitute voltage-depend ion-permeable channels (Thomma et al. (2002)Planta 216:193-202).

Another model is based on the theory that many anti-pathogenic proteinsproduce their action not only by means of the permeabilization of thecitoplasmatic membrane but also by means of citoplasmatic targets, theseproteins once inside the target cell affect the deoxyribonucleic acidsynthesis (DNA), ribonucleic acid (RNA) and proteins. This suggests thatthe ability of the cationic proteins to cause permeabilization of thecitoplasmatic membrane could not be the main cause in the actionmechanism, but a way of search of an intracellular target (Thomma et al.(2003) Curr Drug Targets Infect Disord. 3:1-8).

So far, many types of anti-pathogenic proteins have been identified andis characterized whose possible applications are diverse, because thegenetic engineering provides a resistance strategy to plant diseasesthrough cellular and molecular tools (Thomma et al. (2003) Curr DrugTargets Infect Disord. 3:1-8). It has been demonstrated that theconstitutive expression of radish anti-pathogenic proteins increases thetobacco resistance to the pathogen of leaves Alternaria longipes, (Terraet al. (1995) Plant Cell 7: 573-588), of the same manner occurs intomato plant with Alternaria solani. Also, the constitutive expressionof an anti-pathogenic protein provides a high resistance to the fungusVerticillium dahliae of agronomic importance in the potato crop underfield conditions (Gao et al. (2000) Nat. Biotechnol. 18: 1307-1310).

On the other hand, rice plants expressing the genes of anti-pathogenicproteins of the species Brassica oleracea and B. campestris weremodified to substitute amino acids in different positions, andintroduced individually into rice plants looking resistance toMagnapothe grisea and Xanthomonas oryzae; diseases of great importancein subtropical and tropical countries. These anti-pathogenic proteinsconferred an effective resistance to both disease and the modificationof these genes increased the wide spectrum resistance in transgenic rice(Kawata et al. (2003) JARQ 37: 71-76).

The application of the plant anti-pathogenic proteins as alternative toreduce crop losses due to the attack of pathogen constitutes anadvantage with regard to the application of chemical fungicides. First:plant anti-pathogenic proteins are derived from seeds, roots and tubers,for what they constitute nature substances that are not toxic to thehost plant and neither to people that consume the products from theseplants. Second: as other protein, the anti-pathogenic proteins quicklydegrade like native substances not leaving any residual after theireffectiveness expires (Thomma et al. (2003) Curr Drug Targets InfectDisord. 3:1-8).

The plant anti-pathogenic proteins could also be used for thedevelopment of anti-fungal medications, because the control ofeukaryotic pathogens has always constituted one of the main problems inthe medicine, increasing in the last decades to for the increment ofimmunodepressed patients due to illnesses such as AIDS, cancer andorgans transplant, besides the emergence of multi-drug resistant strainsand the appearance of new species of filamentous fungus as yeasts thatare recognized as opportunist pathogens (Thomma et al. (2003) Curr DrugTargets Infect Disord. 3:1-8).

Due to the similarity between the cells of the mammals and that of thepathogen ones, the anti-fungal compounds should act on molecules thatare not or are rarely present in mammal cells, like components of thecell wall and virulence factors, and they should also be products asmuch natural as possible, with a wide action spectrum, easy to produceand not inducing resistance (Walsh et al. (2000) Medical Mycology, 38:335-347). On the other hand, the fungal cell membranes are attractivetargets for the development of these agents, because the components ofthe fungal membrane like the sphingolipids are structurally different inmammal cells. The plant anti-pathogenic proteins don't have as targetthe biosynthesis of the sphingolipids, but rather they act totally tothe inverse one because their target are their own sphingolipids causingthe permeabilización of the fungal membrane. This provides a highselectivity and therefore, interesting perspectives for the treatment offungal infections. Some plant anti-pathogenic proteins have been foundas: Dm-AMP1, Hs-AFP1 and Rs-AFP2 that are active in micromolarconcentrations against Cándida albicans, a pathogen of great clinicalinterest in humans, which constitutes an example of the potential ofthis type of plant proteins for the development of the therapy (Thommaet al. (2003) Curr Drug Targets Infect Disord. 3:1-8).

An important problem to be solved is to achieve anti-pathogenic productsof protein origin able to efficiently control a wide range of fungal andbacterial pathogens, aspect of a great importance in the agriculture andthe medicine.

DETAILED DESCRIPTION OF THE INVENTION

This invention contributes to solve the problem mentioned above,providing the nucleotide sequence (SEQ ID No. 1) and amino acid sequence(SEQ ID No. 6) of a new anti-pathogenic protein isolated from Nicotianamegalosiphon. The nucleotide sequence of the invention encodes for asmall cysteine rich protein, which has a marked effect over severalpathogenic agents.

It is also object of the present invention, a nucleic acid that encodesfor a polypeptide comprising: a) the identified amino acids sequence asSEQ ID No. 6, or b) an amino acids sequence where one or several aminoacid residues have been eliminated, substituted and added to theidentified sequence of amino acids as SEQ ID No. 6, which maintains itsproperties of controlling the infection by pathogen agents.

In an embodiment of the invention, the gene that encodes for theanti-pathogenic protein of the present invention is used to improve theresistance levels and defense is of plants toward several pathogenagents. Therefore, the present invention includes a method to increasethe resistance to plant diseases caused by pathogenic agents, by meansof the genetic transformation of the plant with a nucleic acid sequence:SEQ ID No. 1 that leads to the constitutive or induced expression of theanti-pathogenic protein: SEQ ID No. 6.

The molecule of nucleic acid object of the present invention can be usedfor the transformation of plants. In a favorite realization, thesequence of DNA that encodes for the anti-pathogenic protein can be usedfor the transformation of the following species of plants: Zea mays,Brassica sp., Medicago sativa, Oryza sativa, bicolor Sorghum, Sorghumvulgare, Pennisetum glaucum, Helianthus annuus, Triticum aestivum,Glycine max, Nicotiana tabacum, Solanum tuberosum, Ipomoea sweetpotatoes, Manihot esculenta, Coffea spp., Coconuts nucifera, Pineapplescomosus, Citrus spp., Theobroma cocoa, Camellia sinensis, Muse spp.,American Persea, Ficus casica, Psidium guajava, Mangifera indicates,Carica papaya, Beta vulgaris, Saccharum spp., Lycopersicon esculentum,Lactuca sativa, Phaseolus vulgaris, Cucumis sativus, Cucumis melo,Hibiscus rosasanensis, Rosa spp., Tulipa spp., Pinus taeda, Pinuselliotii, ponderous Pinus, Pinus contorta, Pinus radiata.

The present invention can be used in a variety of methods in order toobtain plants of agricultural interest that produce the anti-pathogenicprotein. This way, the nucleic acid sequence (SEQ ID No. 1) that encodesfor the anti-pathogenic protein can be used in combination with apromoter that is introduced in a plant of agricultural interest. Aconstitutive promoter can be used in order to expression of high levelsfrom anti-pathogenic protein. In other forms, the sequence that encodesfor the anti-pathogenic protein can be manipulated and fused to aspecific promoter to direct the expression into particular tissue insusceptible plant to a pathogen. Another object of the invention is apolypeptide with the amino acids sequence: SEQ ID No. 6 or SEQ ID No. 7.Anyone of these polypeptides has biological activity on pathogen agents,for what in this invention are denominated anti-pathogenic proteins.Another object of the invention is an amino acids sequence from apolypeptide with at least 60% of homology with the SEQ. ID No.6.

In a preferred embodiment the polypeptides or anti-pathogenic proteinsof the invention are obtained for recombinant way or for chemicalsynthesis. The anti-pathogenic proteins of the invention can beexpressed by DNA recombinant technology in different host systems, andisolated from them. In a materialization of is the invention theanti-pathogenic protein can be expressed in yeasts. In a favouriterealization, the expression for DNA recombinant way is carried out inPichia pastoris, preferably in the supernatant from culture. Startingfrom the hosts, the polypeptides of the invention can be obtainedapplying the techniques of isolation of proteins. The purificationprocess can be achieved using technical immune enzymatic,chromatographic, the cellular precipitate, and other process knownactually.

Variants of amino acid sequences (SEQ ID No. 6) fused with stabilizerspeptides or that they direct the expression to certain compartments ofthe host, and maintain the biological activity demonstrated for thatmolecule, also they are object of the present invention. An example isfused protein whose sequence appears as SEQ ID No. 7. Fragments of theanti-pathogenic protein identified in the Listing of Sequences like SEQID No. 8 and SEQ ID No. 9 that retain the control activity on pathogenagents, also they are object of the present invention. Another aspect ofthe present invention is a bio-product for the control of pathogenagents identified as SEQ ID No. 6, SEQ ID No. 7 or a polypeptide with atleast 60% of homology with the SEQ. ID No. 6.

In this invention these anti-pathogenic proteins are used, for the firsttime, in bio-product that confer high protection levels on the mainplant diseases that taken place by pathogen agents, with high stabilityand low contamination, for what their use presents better publicperception and less regulatory requirements. The bio-product thatcontains the anti-pathogenic protein of the present invention produceshigh protection levels on fungus and bacteria, not reported previously.To achieve the bio-product, the anti-pathogenic protein can formulatethrough a suspension, solution, emulsion, powder, granule, emulsifiableconcentrate, aerosol, impregnated, adjuvant granule, pastures or throughcapsulations. In a realization of the invention, the bio-productcontains the purified polypeptide from a host transformed genetically,or it is used directly contained in the super of a culture of this host.In a favorite realization the host is P. pastoris.

In a materialization of the invention, the anti-pathogenic proteinswhose sequences are claimed can be used, as well as the bio-productsthat contain them, for the first time, for the control of a wide varietyof pathogen as: Aspergillus, Penicilium, Alternaria (Alternariabrassicola; Alternaria solani; Alternaria alternata); Bipolarissacchari; Botrytis cinerea; Cercospora (Cercospora kikuchii; Cercosporazaea-maydis; Cercospora medicaginis; Cercospora sojina; Cercosporasorghi); Cladosporium fulvun; Colletotrichum (Colletotrichumlindemuthianum; Colletotrichum dematium; Colletotrichum graminicola),Diplodia maydis; Erysiphe (Erysiphe graminis f. sp. graminis; Erysiphegraminis f. sp. hordes); Fusarium (Fusarium nivale; Fusarium oxysporum;Fusarium graminearum; Fusarium culmorum; Fusarium solani; Fusariummoniliforme; Fusarium roseum); Helminthosporium (Helminthosporiumturcicum; Helminthosporium carbonum; Helminthosporium maydis);Maganaporthe grisea; Mycosphaerella figensis; Peronospora (Peronosporamanshurica; Peronospora tabacina); Phoma betae; Phytophthora(Phytophthora cinnamomi; Phytophthora cactorum; Phytophthora phaseoli;Phytophthora parasitica; Phytophthora citrophthora, Phytophthoramegasperma f. sp. sojae; Phytophthora infestans), Puccinia (Pucciniasorghi; Puccinia striiformis; Puccinia graminis f. sp. tritici; Pucciniaasparagi; Puccinia recondita; Puccinia arachidis; Pucciniamelanocephala), Pythium (Pythium aphanidermatum; Pythium ultimum);Pyricularia oryzae; Rhizoctonia (Rhizoctonia solani; Rhizoctoniacerealis); Scerotium rolfsii; Sclerotinia sclerotiorum; Septoria(Septoria lycopersici; Septoria glycines; Septoria nodorum; Septoriatritici); Thielaviopsis basicola; Ustilago (Ustilago maydis; Ustilagoscitaminea); Verticillium (Verticillium dahliae; Verticilliumalboatrum); Pseudomonas syringae p.v. glycinea; Xanthomonas campestrisp.v. phaseoli; Xanthomonas campestris p.v. alfalfae; Xanthomonascampestris p.v. translucens; Pseudomonas syringae p.v. syringae; Erwiniacarotovorum p.v. carotovora; Erwinia stewartii; Clavibacter michiganensesubsp. Nebraskense; Pseudomonas avenae; Erwinia chrysanthemi p.v. zea;Erwinia carotovora; Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis y Pseudomonas avenae. In a favorite realization thebioproducto is useful for the control of plant fungi. In amaterialization of the invention the polypeptide included into thebio-product in the concentration range among 1 to 9 μg/ml.

It is also part of the present invention a method for the control ofpathogen agents of plants that is characterized by the application ofthe bio-product to the plants which comprises an identified polypeptidewith SEQ ID No. 6, SEQ ID No. 7 or a polypeptide with at least 60%homology with the SEQ. ID No. 6. In another materialization of theinvention, the method for the control of pathogen agents of plants ischaracterized by the application of the bio-product of the invention incombination with bio-pesticides. Genetically modified plants with thesequence of nucleic acid: SEQ ID No. 1, or a nucleic acid sequence fromSEQ ID No. 1 (transgenic plants), to increase the resistance to plantdiseases produced by is pathogen agents, are also part of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Cloning strategy of interest antipathogenic protein in theexpression vectors in plants (FIG. 1A) and yeast (FIG. 1B).

FIG. 2. Expression of the antipathogenic protein in Pichia pastoris,using the pPIC9K vector, fused to the signal peptide from theSaccharomyces cerevisiae alpha factor. Production of the antipathogenicprotein in the supernatant of P. pastoris (A) and in the purifiedfraction (B).

FIG. 3. Experiment of constitutive expression of the antipathogenicprotein in transgenic plants and evaluation of the disease resistance.The figure represents the percentage of leaves and stems with diseasesymptoms in the controls samples and the transgenic clones expressingthe antipathogenic protein. (FIG. 3A) Effect of Peronospora hyoscyami f.sp tabacina on tobacco leaves. (FIG. 3B) Effect of Alternate solani onpotato leaves. (FIG. 3C) Effect of Phytophthora parasitica on tobaccostems. (FIG. 3D) Effect of Phytophthora infestans on potato leaves. Inthe figures the bars represent: a. inoculated control, b. not inoculatedcontrol, c. clone 1.1, d. clone 1.2, e. clone 1.3.

FIG. 4. Experiment of activity of the antipathogenic protein on severalplant pathogens to different concentrations. The graph represents thepercentage of inhibition of the growth in liquid medium from thepathogen handled to different concentrations of the antipathogenicprotein fused to the signal peptide of the alpha factor (A) andsynthesized without the signal peptide (B). Legend of the FIG. 4B: □effect of the protein fused to the signal peptide of the alpha factor onPhytophthora infestans, ♦ effect of the protein without the signalpeptide on Phytophthora infestans, ▪ control.

FIG. 5. Experiment of the control effect on soil (A) and air (B)pathogens of the formulation of the antipathogenic protein NmDef-02. Inthe figure the bars represent: ▪ control plants, □ plants treated withfungicides, ♦ plant treated with the formulation to of theantipathogenic protein.

FIG. 6. Biological activity of fragments of the antipathogenic proteinNmDef-02 on Phytophthora infestans. □ effect of the peptide SEQ ID No 8on Phytophthora infestans, ♦ effect of the peptide SEQ ID No 9 onPhytophthora infestans.

EXAMPLES Example 1 Preparation of the vegetable material, isolation andcloning of the DNA that encodes for the antipathogenic protein NmDef-02from Nicotiana megalosiphon

The N. megalosiphon specie was grow in pots of 6 inches that containedblack crowd and shell of rice in a proportion (4:1) and maintained ingreen house condition at 23° C. An isolate of Peronospora hyoscyami f.sp. tabacina collected of a tobacco field in Havana was used in theinoculations. The inoculations were carried out in plants of this speciewith 6 weeks of age, placing several drops of 10 μl with a concentrationof 5×10³ spores per ml. The plants were placed in black plastic bagswith high humidity (the humidity was achieved atomizing water inside thebags) during a period of 12 hours, to promote the infection.

The total RNA was extracted of leaves from N. megalosiphon inoculated 6days later, using the system of extraction of total RNA for spin(Promega, Madison, Wis., USA). Finally, the cDNA double chain wassynthesized (cDNA), using the system of synthesis of cDNA from Promega.

The cDNA library was made using subtractive hybridization using thesystem of suppression subtractive hybridization selective (Clontech,Palo Alto, AC, USA). The cDNA obtained from N. megalosiphon plantsinoculated with P. hyoscyami and harvested 6 days after the inoculation,was used as sample for the subtraction. The subtractive library wascloned in the pGEM-T Easy vector (Promega) according to theinstructions.

The sequencing of the cDNA was developed using an automatic sequencer.After the analysis of the sequences, a DNA sequence was selected thathad low homology levels with proteins reported in databases, which wasused in the later experiments.

Example 2 Plant Transformation with the Gene of the AntipathogenicProtein NmDef-02

Production of Tobacco Transgenic Plants: Construction of the BinaryVectors.

In this experiment the complete cDNA of the gene that encodes for theantipathogenic protein was isolated with the oligo nucleotides SEQ IDNo. 2 and SEQ ID No. 3 and cloned in the transformation vector “pCambia2300” in the restriction sites Hind III/Pst I (FIG. 1A). The genetictransformation of plants of Nicotiana tabacum was carried out by themethod from Zambryski et al. (1983) EMBO Journal, 2: 2143-2150. For thisproposal, the strain AT 2260 of Agrobacterium tumefaciens was using themethod of the liquid nitrogen (Hofgen and Willmitzer (1988) Nucl. AcidsRes. 16: 9877) with the developed binary vector. Leaves disks of N.tabacum plants of the variety Petit Havana SR 1 cultivated in vitro weretransformed. Kanamycin was used to 100 mg/L as marker agent. The disksleaves were co-cultivated with the recombinant Agrobacterium for 48hours in Murashige and Skoog (MS) liquid medium. The tobacco plantregeneration (4-6 weeks) was made on MS medium that contained: sucrose25 g/L, 6-Bencil amino purine (BAP) 1 mg/L, acetic naftalen acid (ANA)0.1 mg/L, kanamycin 100 mg/L and claforan (Claf) 500 mg/L. The plantrooting (1-3 weeks) carried out on MS that contained: sucrose 30 g/L,kanamycin 100 mg/L and Claf 500 mg/L.

Production of Potato Transgenic Plants: Construction of the BinaryVectors

In this experiment the complete cDNA of the gene that encodes for theantipathogenic protein was isolated with the oligo nucleotides of theSEQ ID No. 2 and SEQ ID No. 3 and cloned in the transformation vector“pCambia 3300” in the restriction sites Hind III/Pst I (FIG. 1A). Thevegetable material that was used in the experiments of tissue cultureand transformation was taken of in vitro plants from the cultivar ofpotato “Desiree.” The plants were grown in test tubes on MS medium. ThepH of the culture medium was adjusted at 5.7. Plants of four weeks ofcultivation were used, maintained in rooms with 25° C. and anillumination of 2000 Lux. The culture medium that served as base for theregeneration experiments and transformation were the SC (MS salts,vitamin B1 0.4 mg/L, myo-inositole 100 mg/L, sucrose 20 g/L, BAP 3.5mg/L, ANA 0.01 mg/L, phytoagar 6 g/L), the SB (MS salts, myo-inositole100 mg/L, sucrose 20 g/L, AG₃ 3.5 mg/L, phytoagar 6 g/L) and the PP (MSsalts, vitamin B1 0.4 mg/L, myo-inositole 100 mg/L, pantothenate ofcalcium 2 mg/L, sucrose 30 g/L, phytoagar 6 g/L, activated carbon 5 g/Land Nitrate of Silver-thiosulfate of Sodium 1 mg/L (STS).

For the transformation the strains of Agrobacterium “At2260” and “LBA4404” were used. The bacteria was cultivated in a culture medium withyeast extract 1 g/L, bacto-peptone 1 g/L, sucrose 5 g/L and Lab-lemcopowdered 5 g/L, to 28° C. in the dark condition until reached an opticaldensity (DO)₆₂₀ 0.7-0.9.

The procedure of transformation-regeneration was developed in twostages, in the is following way: segments of stems of in vitro plants of4 weeks of cultivation were incubated on MS medium during 12-16 hours at25° C. in dark, then the infection with A. tumefaciens was developed bymeans of the incubation of the explantes during 7 minutes with 1 mL ofthe bacterial suspension for each 20 ml of MS medium; the explantes wereco-cultivated on SC medium during 48 hours at 22° C. in dark and theexplantes was placed on a sterile filter paper. The washing of theexplantes on MS medium and drying with sterile filter paper was carriedout carefully. In a first stage they were cultivated during 15 days onlight condition on selective medium SC, 500 mg/L of claforan and 5 mg/LPhosphinothricin (PPT) and in a second stage they were cultivated onlight condition on selective medium SB, 500 mg/L of claforan and 5 mg/LPPT. Finally the small plants were individualized on selective mediumPP, with 500 mg/L of claforan and selected in 5 mg/L of PPT.

Example 3 Evaluation of the Effect of the Antipathogenic ProteinNmDef-02 on the Disease Resistance

Experiment of Tobacco Disease Resistance to Peronospora hyoscyami f. sp.Tabacina.

Plants with roots, resistance to the kanamycin antibiotic and with thegene of the antipathogenic protein were planted in pot for itsadaptation in green house condition during 45 days. After that period agroup of 100 transgenic clones of 6 weeks old were inoculated with asuspension of P. hyoscyami f. sp. tabacina placing several drops of 10μl with a concentration of 5×10³ spores/ml. The plants were placed inblack plastic bags and spraying with water during a period of 12 hours,to promote the infection. The evaluation of the susceptibility wascarried out measuring the percentage of leaves with symptoms of thedisease one week later (FIG. 3A). As it can be appreciated in the FIG.3A high resistance levels were achieved to this pathogen, when wecompare the percentage of leaves with symptoms between the inoculatedcontrol and the different clones, which shows the utility of theantipathogenic protein used for the control of this important pathogen.

Experiment of Tobacco Disease Resistance to Phytophthora parasitica.

Plants with roots, resistance to the kanamycin antibiotic and with thegene of the antipathogenic protein were planted in pot for itsadaptation in green house during 45 days. After that period a group of100 transgenic clones of 6 weeks old were inoculated with P. parasiticaand the percentage of stem with disease symptoms was evaluated one monthlater.

For the evaluation procedure the pathogen P. parasitica was grown inPetri plate that contained Potato Dextrose Agar medium (PDA) at aconcentration of 39 g/L. The pathogen was incubated at 27° C., during 10days. For the inoculation of tobacco plants a PDA disk (diameter=1 cm)with the pathogen was placed in the base of the stem and incubated at28° C., with high humidity FIG. 3B). As it can be appreciated in theFIG. 3B the clones expressing the antipathogenic protein achieved highresistance levels never seen before to this soil pathogen that causesimportant losses in the seedling phase of this crop. This resultdemonstrates that it is feasible the use of this protein for the controlof this pathogen.

Experiment of Potato Disease Resistance to Phytophthora infestans

This test consisted in inoculation of Phytophthora infestans in potatotransgenic plants of 5 weeks old under controlled conditions of light,temperature and relative humidity. About 100 clones of 5 weeks old thatexpress the antipathogenic protein were spreading, with a suspension of10⁶ zoospores/ml. The clones were maintained under controlled conditionswith a relative humidity between 85-95% and a temperature of 23° C. Thepercentage of leaves with symptoms was used as measure of susceptibilityto the pathogen one week after the inoculations (FIG. 3C). This was anunexpected result, since this pathogen is extremely difficult tocontrol. In the FIG. 3C, the clones expressing the antipathogenicprotein showed high resistance levels, compared with the controls. Thisresult points out the potential for the use of this protein in thecontrol of this pathogen in particular, for its importance at worldlevel.

Experiment of Potato Disease Resistance to Alternaria solani

The fungus Alternaria solani was inoculated in 100 potato transgenicplants of 5 weeks old under controlled conditions of light, temperatureand relative humidity. The clones were spreading with a suspension of10⁶ spores/ml. The clones were maintained under controlled conditionswith a relative humidity between 85-95% and a temperature of 20° C. Thepercentage of leaves with symptoms was used as measure of susceptibilityto the pathogen one week after the inoculations (FIG. 3D). The threeanalyzed clones showed high resistance levels to this pathogen, whichfor the first time offers a potential for the use of this protein forthe control.

Example 4 Construction of the Expression Vector of the AntipathogenicProtein NmDef-02, in an Extracellular Way, in the Super of Pichiapastoris Culture

The gene that encodes for the antipathogenic protein from N.megalosiphon was isolated using the specific oligo nucleotidescorresponding to the SEQ ID No. 4 and SEQ ID No. 5 to obtain thecomplete sequence of the gene that encodes for the antipathogenicprotein NmDef-02, with the enzymatic restriction sites Xho I/EcoR I,necessary for the cloning in the expression vector pPIC9k. This cloningstrategy adds to the protein of interest in the end amino-terminal thesignal peptide of the factor alpha of Saccharomyces cerevisiae (FIG.1B), for what the resulting protein belongs to the SEQ ID No. 7. Theplasmid was linearized with Bgl II before transforming the strain GS115of P. pastoris. The transformation was carried out by electroporation.The strain GS115 is a mutant auxotrophic his3 which acquires a phenotypeHis+ after the transformation. The clones His+ was selected on minimumglucose medium; the clones were cultivated on minimum glycerol mediumand induced with methanol during 126 hours at 28° C.

The transformed clones were identified by Dot-Blot. Using the techniqueof Southern Blot was determined in which the integration had happenedfor substitution of the gene AOX1 from P. pastoris for the expression ofrecombinant plasmid, which is correspondence with a phenotype Mut^(s)(low methanol) and His⁺ . P. pastoris secrets low levels of own proteinsand its culture medium doesn't need proteins supplements, for can behope that a protein that is secreted to the subcellular medium, whichconstitute most of the total of proteins in the culture (more than 80%)(Tschopp et al. (1987) Bio/Technology 5:1305-1308). The expression ofthe antipathogenic protein in P. pastoris was carried out in bash of 5litres by addition of the methanol to the culture medium. The expressionof the antipathogenic protein and its integrity were checked by massspectrophotometry.

Example 5 Purification and Assay of the Biological Activity of theAntipathogenic to Protein

The antipathogenic protein fused to the signal peptide of the alphafactor (NmDef-Plus) was purified from the supernatant of the culturemedium, by dialysis in 25 mm of acetate of sodium to pH 4.5; the productof the dialysis spent through a resin of cationic exchange CM-Sepharosabalanced Fast-flow with 25 mm of acetate of sodium, pH 4.5; and proteinswere eluted with 1 M sodium chloride, 50 mm of Tris pH 7.6. Thefractions that contained the protein were collected and concentratedusing an ultra-spin system with a membrane with a pores size (cut-off)of 3 kDa. For the detection a wavelength of 254 nm was used. Thepurification was checked by electrophoresis in polyacrylamide gel withSDS-PAGE, Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis(15% ris-Glicne) and the proteins were visualized by silver staining(FIG. 2).

The antifungal activity of the antipathogenic protein was quantifiedthrough the spectrophotometry method and the analysis of the mycelium bystaining with blue lactophenol through optic microscopy (Terras et al.(1992) J. Biol. Chem. 267: 14301-15309). The evaluation was carried outin 96 wells-plates, in which 50 μL of potato-glucose liquid medium, 50μL of spores suspension of the pathogen and 20 μL of the antipathogenicprotein partially purified were added.

The percentage of inhibition of the growth (PIC) of the pathogen wasdetermined according to that reported previously (Terras et al. (1992)J. Biol. Chem. 267: 14301-15309), by means of the following equation:

${PIC} = {\frac{{{DO}_{595\mspace{14mu}{nm}}\mspace{20mu}{control}} - {{DO}_{{595\mspace{14mu}{nm}}\mspace{20mu}}{treatment}}}{{DO}_{595\mspace{14mu}{nm}}\mspace{14mu}{control}} \times 100}$

The relation among inhibition percentages comes from the application ofthe former equation to the realized readings (to 595 nm) of the controlsand the treated samples 48 hours after the assay was initiate (FIG. 4A).

The antipathogenic protein without the signal peptide of the alphafactor was chemically synthesized, and its effect was evaluated on thepathogen P. infestans to different concentrations according to previousreport (Terras et al. (1992) J. Biol. Chem. 267: 14301-15309) (FIG. 4B).

In FIGS. 4A and 4B, the antipathogenic protein achieved high levels ofinhibition growth of plants pathogens, either fused to the signalpeptide of the alpha factor or to chemically synthesized without thesignal peptide. It was very interesting to observe, for the first time,the inhibition of important pathogens of plants, not achieved until themoment by other antifungal proteins reported. Another non prospectiveresult and that allows the employment of this antipathogenic proteinwith a wide action spectrum, were the high inhibition levels that showedin bacterial pathogen (Table 1).

TABLE 1 Effect of the antipathogenic protein NmDef-02 on bacterialpathogens. Bacteria Species IC₅₀ (μM) Xanthomonas campestris p.v.phaseoli 40 ± 3 Pseudomonas syringae p.v. syringae 26 ± 6 Erwiniacarotovorum p.v. carotovora 86 ± 8 Pseudomonas avenae 17 ± 5 Erwiniachrysanthemi p.v. zea 11 ± 8 IC₅₀: concentration of NmDef-02, where thebacteria growth is inhibit in a 50%. ± Media standard deviation.

Example 6 Demonstration of the Control of a Formulation of theAntipathogenic Protein NmDef-02 on Soil and Air Fungal Pathogen

Seeds of the N. tabacum species were treated with the formulation of theantipathogenic protein (9 μg/ml) and 5% of sodium alginate, after thatthey were germinated in 6 inches pots that contained black crowd andrice in a proportion (4:1) and maintained in greenhouse at 23° C. Ascontrols treatments were used seed treated with 10% of hypochlorite ofsodium and others without any treatment type. The evaluations werecarried out at 30 days and the percentage of dead plants due to naturalinfection was determined (FIG. 5A).

On the other hand, 100 plants in greenhouse were spraying with theformulation of the antipathogenic protein with a concentration of theactive principle of 5 μg/ml and 5% sodium alginate. For controls,aspersions with the fungicidal Benomil at concentration of 40 g/L andwater were used. The percentage of leaves with symptoms was evaluatedthrough of the natural infection, at 45 days after the application oftreatments (FIG. 5B).

In the FIGS. 5A and 5B the formulation of the antipathogenic proteinachieved the control of soil and air fungal pathogen in comparison withthe controls used; this result has not been reported until the moment,allowing the use of this protein as a bioproduct in the control of plantpathogens.

Example 7 Biological Activity of Fragments of the Antipathogenic ProteinNmDef-02

Fragments of the antipathogenic protein NmDef-02 were obtained bychemical synthesis (SEQ ID No. 8 and SEQ ID No. 9). The effect on thepathogen P. infestans was evaluated to different concentrationsaccording to the information reported (Terras et al. (1992) J. Biol.Chem. 267: 14301-15309), which is shown in FIG. 6. The obtained resultallows the use of fractions of the protein to achieve the control orinhibition of pathogen when the fraction of the protein of the SEQ IDNo. 9 is used.

Incorporation Of Sequence Listing

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “sequence_listing.txt”,created on Apr. 17, 2013. The sequence_listing.txt file is 4 kb in size.

The invention claimed is:
 1. A method for the control of pathogen agentsof plants said method comprising application of a bioproduct thatcomprises the polypeptide identified as SEQ ID NO: 6 or SEQ ID NO: 7 tothe plants.
 2. The method according to claim 1, wherein the polypeptideis purified from a genetically transformed host or it is used directlyas contained in a supernatant of a culture of such a host.
 3. The methodaccording to claim 2, wherein the host is P. pastoris.
 4. The methodaccording to claim 1, wherein the pathogen agent is a fungal pathogen.5. The method according to claim 1, wherein the polypeptide is in aconcentration range between 1 to 9 μg/ml.
 6. A method for the control ofpathogen agents of plants said method comprising application of abioproduct that comprises the polypeptide identified as SEQ ID NO: 6 orSEQ ID NO: 7 mixed with biopesticides to the plants.